Project Summary The human colon is a remarkable organ, playing critical roles in drug uptake and metabolism as well as harboring the 100 trillion microbial cells of the microbiome, which itself has multiple impacts on human health. For these reasons, there is a widespread need in academia and the biotechnology marketplace for in vitro studies of human colon physiology and the interaction between colon tissue and the anaerobic bacteria of the microbiome. To meet this need, Altis Biosystems, Inc., an early stage biotechnology company, has collaborated with scientists at the University of North Carolina (UNC) at Chapel Hill to develop a new platform for cell culture to co-culture normal, human colonic epithelial cells with anaerobic microbiota, named the Self-sustaining Intestinal Microbiome Platform (SIMPle). SIMPle is an easy-to-use and intuitive platform to replicate the steep oxygen gradient across the in vivo colonic epithelium, thus create the appropriate environment required for anaerobes while maintaining viable, healthy epithelial tissue. We have finished the SBIR Phase I program by designing, prototyping, validating the SIMPLe that fits a standard 12-well plate (SIMPle12). The generation of a steep O2 gradient was confirmed, and its impact on cells was characterized. The co-culture of facultative anaerobes with human colonic epithelium in SIMPle12 platforms was validated. All proposed milestones in the Phase I SBIR were accomplished, thus providing a solid foundation for this Phase II SBIR application. The focus of this Phase II proposal is to scale up SIMPLe platform to a 96-well format (SIMPle96) to meet the market needs for screening assays. The SIMPle96 platform will be prototyped and validated, and human colonic epithelium and co-culture with obligate anaerobes will be characterized. Use SIMPle96 platform for screening probiotics will be demonstrated. An external validation of the platform will be performed to demonstrate its utility in microbe-intestinal assays. The SIMPle co-culture platforms are expected to revolutionize microbiome research by enabling the function of host-microbe interactions to be interrogated.